The agricultural and pharmaceutical industry seeks production of compounds in high yield, and when a compound contains one or more chiral centers, it is often desirable to produce a single isomer. The products of the present invention are useful as precursors for chemicals of high value in these industries. Specifically, cis-4-hydroxy-D-proline (CHDP) and trans-4-hydroxy-L-proline (THLP) are useful for preparing agrochemicals and pharmaceuticals.
CHDP is prepared commercially by the chemical epimerization of THLP (Greenstein, J. P. and Winitz, M., Chemistry of the Amino Acids, vol.3, chapter 29, John Wiley and Sons: New York (1961)). Required for the synthesis of CHDP, THLP is prepared commercially from hydrolyzed animal gelatin, which contains approximately 13% of the desired amino acid (U.S. Pat. No. 3,860,607). Chemically epimerizing THLP to CHDP uses either basic reaction conditions (e.g., barium hydroxide under pressure at 200.degree. C. for 6 h) or acidic reaction conditions (e.g., refluxing acetic acid/acetic anhydride). Producing mixtures of THLP and CHDP by chemical epimerization typically (1) uses high temperatures, (2) requires separating the base or acid from the desired hydroxyproline diastereomers before separating CHDP from THLP, and (3) produces highly caustic or acidic waste streams. These conditions introduce undesirable cost and handling risks.
Enzymes, as opposed to harsh chemicals, can also be used in epimerization reactions. Reactions carried out with the aid of enzymes or intact microorganisms have been used with increasing frequency and increasing success to catalyze synthetic chemical reactions. Biological processes are commonly perceived as being less harmful to the environment than chemical manufacturing processes. Amino acid racemases catalyze formation of a racemic mixture from either the D or L form of the free amino acid by equilibrating configuration at the .alpha.-carbon. Amino acid epimerases also catalyze equilibration of configuration at the .alpha.-carbon. However, since epimerases act on compounds possessing an additional asymmetric carbon, a diastereomer of the substrate is formed rather than its antipode.
4-Hydroxyproline epimerase (EC 5.1.1.8, also known as hydroxyproline 2-epimerase) catalyzes the conversion of THLP to CHDP and CHDP to THLP. The enzyme was the first amino acid-racemizing enzyme to be characterized in an essentially homogenous state, permitting direct investigation of cofactor status (Adams et al., J. Biol. Chem. 239:1525-1535 (1964)). An approximately equimolar mixture of THLP and CHDP is obtained when starting with either diastereomer. The enzyme also epimerizes cis-4-hydroxy-L-proline (CHLP) or trans-4-hydroxy-D-proline (THDP) to a mixture of CHLP and THDP. 3-Hydroxyprolines have also been reported to be substrates for the enzyme. ##STR1##
4-Hydroxyproline epimerase has been isolated from Pseudomonas putida (Finlay et al., J. Biol. Chem. 245:5248-5260 (1970)), Pseudomonas striata (Adams et al., J. Biol. Chem. 239:1525-1535 (1964)), Pseudomonas fluorescence (Ito et al., Anal. Biochem. 151:510-514 (1985)), Pseudomonas aeruginosa (Manoharan et al., J. Biosci. 2:107-120 (1980)), Pseudomonas ovalis and Alcaligenes sp. (Drawert et al., Chem., Mikrobiol., Technol. Lebensm. 5:165-169 (1978)), Achromobacter (Jayaraman et al., Indian J Biochem. 2:153 (1965)), and Aerobacter aerogenes (Adams et al., Methods Enzymol. 17B:266-306 (1971)). In addition, the enzyme has been isolated from a fermentation of Pseudomonas No. 109 which was cultured in the presence of 4-hydroxy-L-proline (JP 60221083).
Although these sources of 4-hydroxyproline epimerase are known, a novel and enhanced source for this biocatalyst would be useful to industry in the production of high value agrochemicals and pharmaceuticals.